Our Specific aims continue to explore the biology of stem cells. Specific aim 1 uses simplified gradient separation to isolate stem cells. We plan studies which will isolate stem cells by functional characteristics. We believe that the most primitive quiescent stem cell may not express cell surface antigens attributable to long term engrafting cells. We have evidence that mul tipotential stem cells can proliferate and differentiate into cells of different germ layers. We plan additional clonal a nalysis to evaluate the engraftment capacity of pure stem cell populations. We will examine the nonhematopoieitc (epithelial) engraftment of our enriched populations of marrow derived stem cells. We plan to examine the factors which are known to regulate the proliferation and differentiation of mouse adult and embryonic stem cell populations. Finally, in specific aim 1, we will determine if stem cells isolated by gradient separation technology have the capacity to convert marrow cells into epithelial tissues in response to injury signals. In the second aim, importantly, we will attempt to distinguish between the presence of tissue specific stem cells and the existence of a pluripotent stem cell which resides in the bone marrow which, when stimulated by external injury signals, will respond by mobilization to the site of injury, convert into a new germ layer, and aid in repair of the injury. Relevance Bone marrow transplantation has been utilized for hematological failure or malignancy with increasing success. Recent animal models and a few clinical trials have used stem cell populations to repair injury via transplantation. We are attempting to define the cell types which can be used for such repair, develop simple and universal methods for isolating those cells and determining what factors in the microenvironment are responsible for stem cell self renewal and differentiation.